2´-O-Methylation of Capped RNA (M0366)

Introduction

This
protocol is designed to methylate up to 10 μg of capped RNA in a 20 μl reaction.
Reaction size can be scaled up as needed.

Protocol

Combine capped RNA and nuclease-free water
in a final volume of 16 μl. (Refer to step 1 in the notes on
use).

Heat at 65°C for 5 minutes (Refer to step
2 in the notes on use).

Place tube on ice for 5
minutes.

Add the following components in the order
specified:

Denatured capped RNA (from above)

16.0 μl

10X Capping Buffer

2.0 μl

SAM (4 mM, dilute 32 mM stock to 4 mM)

1.0 μl

mRNA Cap 2´-O-Methyltransferase (50 U/μl)

1.0 μl

20.0
μl

Note: Use of RNase Inhibitor is recommended to
enhance stability of RNA in the reaction. Add 0.5 μl of RNase Inhibitor (e.g.,
Murine RNase Inhibitor NEB #M0314 ) during
reaction set up. Subtract the additional volume from the amount of
H2O used in the reaction.

Incubate at 37°C for 60 minutes (For RNA
less than 200 nt long increase incubation time to 2 hours).

Proceed with purification of the RNA (if
required) for downstream applications.